Provided herein are methods for introducing one or more fluorine atoms, particularly radioactive fluorine atoms, onto biomolecules that include at least one thiol group, such as polypeptides having at least one thiol group. Further provided are methods for preparing linkers that may be employed to introduce one or more fluorine atoms onto such biomolecules. Also provided are bioconjugates and fluorinated polypeptides produced using the disclosed methods and agents.
Because radioactive fluorine atoms, such as F-18, have a relatively short lifetime of about 110 minutes, highly efficient methods are required for site-specifically introducing the radiofluorine onto biomolecules. Previous efforts involved reaction of an aldehyde-reactive functional group with a radiofluorine-substituted aldehyde, followed by purification, and then reaction with the biomolecule having at least one thiol group. Site-selective addition becomes increasingly challenging as the number of amino acid residues present in the target increases. Typical methods of labeling such polypeptides, particularly with radiofluorine atoms utilize non-specific reactions, such as reactions based on use of N-hydroxysuccinimide activated ester chemistry, generating heterogeneous products.
In alternative approaches, aldehyde-reactive functional groups (e.g., aminoxy) are introduced during solid phase resin synthesis of peptides by protecting all reactive groups present on the polypeptide other than the target group, requiring multiple reaction and purification steps. Moreover, these protection-deprotection approaches are not readily applicable to biologically produced polypeptides.
Therefore, there is a continuing need for efficient and site-specific methods for introducing fluorine atom(s) including radioactive fluorine atom(s) onto biomolecules, such as polypeptides.